LOCUS       sequence-333999-        9353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculoviral transfer vector to express nsp12-His6-3xFlag 
            (SARS-CoV-2) in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9353)
  AUTHORS   Bertolin AP, Weissmann F, Zeng J, Posse V, Milligan JC, Canal B, 
            Ulferts R, Wu M, Drury LS, Howell M, Beale R, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2425-2443. doi: 10.1042/BCJ20210200.
  PUBMED    34198323
REFERENCE   2  (bases 1 to 9353)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Biochem J"; date: "2021-07-16- 16"; volume: "478"; issue: "13"; 
            pages: "2425-2443"
COMMENT     Sequence Label: pBIG1a_nsp12-His6-3xFlag (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..9353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(347..352,353..369,370..375))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(771..804)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(850..869)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    894..942
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        1037..1128
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     1053..1070
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             3986..4003
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4010..4075
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4208..4342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4245..4264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4299..4318
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4657..4822
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     CDS             5346..6137
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      6178..6632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6265..6284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6474..6495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6658..6762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6763..6831,6832..7623)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6981..7000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7794..8382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8283..8302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8536..8553
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  8687..8911
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(join(8978..9353,1..158))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg
       61 ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga
      121 ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc ataacatcaa
      181 acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag actgtacaaa
      241 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
      301 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt tacgaaccga
      361 acaggcttat gtcaactggg tttgtgcctt catccgtttc cacggtgtgc gtcacccggc
      421 aaccttgggc agcagcgaag tcgaggcatt tctgtcctgg ctggcgaacg agcgcaaggt
      481 ttcggtctcc acgcatcgtc aggcattggc ggccttgctg ttcttctacg gcaaggtgct
      541 gtgcacggat ctgcccttgc ttcaggagat cggtagacct cggccgtcgc ggcgcttgcc
      601 ggtggtgctg accccggatg aagtggttcg catcctcggt tttctggaag gcgagcatcg
      661 tttgttcgcc caggactcta gctatagttc tagtggttgg ctacagcttt gtttgtacta
      721 tcaacaggtt gaactgctga tcaacagatc ctctacgcgg ccgcggtacc ataacttcgt
      781 atagcataca ttatacgaag ttatctgggt ttaaacgctc tatggtctaa agtttttatc
      841 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
      901 aaaaagacag aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt
      961 cccagggctg gcactctgtc aagtgaacgc tctatggtct aaagatttaa atcgacctac
     1021 tccggaatat taatagatca tggagataat taaaatgata accatctcgc aaataaataa
     1081 gtattttact gttttcgtaa cagttttgta ataaaaaaac ctataaatat tccggattat
     1141 tcataccgtc ccaccatcgg gcgcggatcc agccaccatg tctgctgacg ctcagtcctt
     1201 cctgaaccgt gtgtgcggtg tctctgctgc tcgtctgact ccttgcggta ctggaacctc
     1261 taccgacgtg gtgtaccgtg ctttcgacat ctacaacgac aaggtggccg gtttcgctaa
     1321 gttcctcaag accaactgct gccgcttcca agagaaggac gaggacgaca acctgatcga
     1381 ctcctacttc gtggtcaagc gtcacacctt ctccaactac cagcacgagg aaactatcta
     1441 caacctgctg aaggactgcc ccgctgtggc taagcacgac ttcttcaagt tccgtatcga
     1501 cggcgacatg gtgccccaca tctctcgtca gcgtctgacc aagtacacca tggctgacct
     1561 ggtgtacgct ctgcgccact tcgatgaggg aaactgcgac accctgaaag agatcctggt
     1621 cacctacaac tgctgtgacg acgactactt caacaagaag gattggtacg acttcgtcga
     1681 gaaccccgac atcctgcgtg tgtacgctaa cctgggagag cgtgtgcgtc aggctctgct
     1741 caagactgtg cagttctgcg acgctatgcg taacgctggt atcgtgggag tgctgaccct
     1801 ggacaaccag gacctgaacg gcaattggta tgatttcggc gacttcatcc agactacccc
     1861 tggttctggt gtccccgtgg tggactctta ctactccctg ctgatgccca tcctgactct
     1921 gacccgtgct ctgaccgctg agtctcacgt ggacaccgac ctgaccaagc cttacatcaa
     1981 gtgggacttg ctgaagtacg atttcaccga ggaacgcctg aagctgttcg accgttactt
     2041 caagtactgg gaccagacct accatcctaa ctgcgtgaac tgcctggacg accgttgcat
     2101 cctgcactgc gctaacttca acgtgctgtt ctccactgtg ttccctccaa cctccttcgg
     2161 tcctctcgtg cgcaagatct tcgtggacgg tgtccctttc gtggtgtcca ccggttacca
     2221 cttccgtgaa ctgggtgtcg tgcataacca ggacgtcaac ctccactcct ctcgtctgtc
     2281 cttcaaagag ctgctggtct acgctgctga ccccgctatg catgctgctt ccggaaacct
     2341 gctgctggac aagcgtacca cttgcttctc cgtggctgcc ctgactaaca acgtggcctt
     2401 ccagaccgtg aagcccggca atttcaacaa ggacttctac gatttcgccg tgtccaaggg
     2461 cttcttcaaa gaaggctcct ccgtcgagct gaagcacttc ttcttcgctc aagacggcaa
     2521 cgctgctatc tccgactacg actactaccg ttacaacttg cccaccatgt gcgacatccg
     2581 tcagctgctg ttcgtggtcg aggtggtgga caagtacttc gactgctacg acggtggttg
     2641 catcaacgcc aatcaagtga tcgtgaacaa cctggacaag tccgctggct tccccttcaa
     2701 caaatggggc aaagctcgtc tgtactacga ctccatgtcc tacgaggacc aggacgctct
     2761 gttcgcttac accaagcgta acgtgatccc caccatcact cagatgaacc tgaaatacgc
     2821 catctccgct aagaaccgtg ctcgtaccgt ggctggtgtc tccatctgct ccaccatgac
     2881 caaccgtcag ttccaccaga agctgctgaa gtcaatcgct gctacccgcg gtgctaccgt
     2941 ggtcatcggt acttccaagt tctacggtgg ctggcacaac atgctgaaaa ccgtgtactc
     3001 cgacgtggaa aaccctcacc tgatgggctg ggactacccc aagtgcgatc gtgctatgcc
     3061 taacatgctg cgtatcatgg cttccctggt gctggctcgc aagcacacta cttgctgctc
     3121 cctgtctcac cgtttctacc gtctggctaa cgagtgcgct caggtgctgt ccgagatggt
     3181 catgtgcggt ggttccctgt acgtgaagcc tggtggtact tcctctggcg acgctaccac
     3241 cgcttacgct aactccgtgt tcaacatctg ccaggctgtg accgctaacg tgaacgctct
     3301 gctgtccacc gacggcaaca agatcgctga caaatacgtg cgtaacctgc agcaccgtct
     3361 gtacgagtgc ctgtaccgta accgtgacgt cgacactgac ttcgtgaacg agttctacgc
     3421 ttacctgcgc aagcacttca gcatgatgat cctgtccgac gacgctgtcg tgtgcttcaa
     3481 ctccacctac gcttcccaag gcctggtggc ttccatcaag aacttcaagt ccgtgctgta
     3541 ctaccagaac aacgtgttca tgtccgaggc taagtgctgg accgagactg acctgactaa
     3601 gggtccccac gagttctgct cccagcacac catgctggtc aagcagggcg acgattacgt
     3661 gtacctgcct tatcctgatc cttctcgtat cctcggcgct ggttgcttcg tggatgacat
     3721 cgtcaagacc gacggaaccc tgatgatcga gcgtttcgtg tccctggcta tcgacgctta
     3781 ccctctgact aagcacccca atcaagagta cgctgacgtg ttccacctgt acctgcagta
     3841 catccgcaag ctgcacgacg agctgaccgg acacatgctg gacatgtact ccgtgatgct
     3901 gaccaacgac aacacctctc gttactggga gcccgaattc tacgaggcta tgtacacccc
     3961 tcacaccgtg ctgcagggtg gctcccacca tcaccaccat cacggttctg actacaagga
     4021 ccacgacggc gattacaagg atcatgacat cgactacaaa gacgacgatg acaagtaaaa
     4081 gcttgtcgag aagtactaga ggatcataat cagccatacc acatttgtag aggttttact
     4141 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     4201 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4261 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4321 tgtatcttat catgtctgga tctgatcact gcttgagcct agaagatccg gctgctaaca
     4381 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta tcataaccat
     4441 ttaaataaat ctatatctca atcggggtta cgtcgatacc ccaccgagac cccattggga
     4501 ccaatacgcc cgcgtttctt ccttttcccc accccaaccc ccaagttcgg gtgaaggccc
     4561 agggctcgca gccaacgtcg gggcggcaag ccctgccata gccactacgg gttaattaaa
     4621 aactatatct caatcggggt ttaaacagga gatccgaacc agataagtga aatctagttc
     4681 caaactattt tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat
     4741 ctattttgtc actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc
     4801 ccaactattt tgtccgccca cagcggggca tttttcttcc tgttatgttt ttaatcaaac
     4861 atcctgccaa ctccatgtga caaaccgtca tcttcggcta ctttttctct gtcacagaat
     4921 gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat atcaacgctt
     4981 atttgcagcc tgaactggga ggcagaataa atgatcatat cgtcaattat tacctccacg
     5041 gggagagcct gagcaaactg gcctcaggca tttgagaagc acacggtcac actgcttccg
     5101 gtagtcaata aaccggtaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg
     5161 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
     5221 gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt
     5281 tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa
     5341 acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
     5401 tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
     5461 atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
     5521 atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
     5581 agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
     5641 ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
     5701 caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
     5761 gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
     5821 aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
     5881 gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
     5941 ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
     6001 agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct
     6061 tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca
     6121 aggtagtcgg caaataatgt ctaacaattc gttcaagccg acggattggc gaatgggacg
     6181 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6241 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6301 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6361 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     6421 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttctta atagtggact
     6481 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6541 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6601 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     6661 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6721 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6781 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6841 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6901 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6961 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7021 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7081 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7141 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     7201 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     7261 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     7321 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     7381 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     7441 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     7501 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7561 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7621 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7681 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7921 gcgcagatac caaatactat tcttctagtg tagccgtagt taggccacca cttcaagaac
     7981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8401 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     8461 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     8521 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     8581 tttctcctta cgcatctgtg cggtatttca caccgcatag accagccgcg taacctggca
     8641 aaatcggtta cggttgagta ataaatggat gccctgcgta agcgggtgtg ggcggacaat
     8701 aaagtcttaa actgaacaaa atagatctaa actatgacaa taaagtctta aactagacag
     8761 aatagttgta aactgaaatc agtccagtta tgctgtgaaa aagcatactg gacttttgtt
     8821 atggctaaag caaactcttc attttctgaa gtgcaaattg cccgtcgtat taaagagggg
     8881 cgtggccaag ggcatggtaa agactatatt cgcggcgttg tgacaattta ccgaacaact
     8941 ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta cttgggtcga
     9001 tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc actgcgggat
     9061 cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg gcctcatgct
     9121 tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg gagactgcga
     9181 gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg taagccgcga
     9241 gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc ttactacgga
     9301 gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct acg
//
